Immunologies! tolerance that is mediated by FoxpS-expressingregulatory T cells has obvious therapeutic triplications for intervention in autoimmunity, transplant rejection and allergy. Recent years have seen considerable progress in understanding the role of antigen-specific receptors on Treg that are involved in ntra- and extra-thymic generation of Treg and their recruitment to specific sites of antigen deposition. The atter facilitates effective suppression of co-recruited effector cells. Antigen-specific Treg are generated when the TCR of developing thymocytes binds to cognate TCR-ligands expressed by thymic epithelial cells, but it is not clear to what extent expression of ligands by cortical epithelial cells and/or cross-presentation by hemopoietic cells contribute to Treg generation. Furthermore, it is unclear whether regulation of expression by the AIRE transcription factor can contribute. With regard to Treg generation by subimmunogenic delivery of TCR-ligands in peripheral lymphoid tissue, it is not clear whether results obtained in TCR transgenic mice can be extrapolated to wt mice and exploited to specifically suppress unwanted immune responses. Finally, there is very little information on molecular mechanisms involved in Treg-mediated suppression in vivo. In order to understand better the intrathymic process of Treg generation and its possible regulation by the AIRE transcription factor, we propose in Aim 1 to analyze the generation of FoxpS-expressingthymocytes with a transgenic T cell receptor (TCR-HA) specific for peptide 107-119 of influenza hemagglutinin (HA) in reaggregate fetal thymic organ cultures (RFTOC) in which cortical and medullary epithelial cells differ in their expression of TCR ligands that are able to induce Treg. In Aim 2 we propose to induce Treg with specificity for foreign ligands with the goal to intervene with transplant rejection, graft versus host disease and allergy by exploring the translation of results obtained in TCR transgenic mice into wt mice. Aim 3 will deal with the correlation of Foxp3 promoter binding and regulated gene expression in Treg as well as gene expression analysis in regulated versus non-regulated CD8+ effector cells with the goal to identify molecular mechanisms that govern Treg-mediated suppression of T effector function.